Abstract
Purpose
Papillary thyroid cancer (PTC) is more common in women than in men. It has been suggested that estrogen may be involved in its development, as has previously been shown for breast, endometrial and ovarian cancer. The purpose of this study was to assess correlations between the expression of the estrogen receptor alpha36 (ERα36) and the glucose regulated proteins GRP78 and GRP94 (chaperones involved in glycoprotein folding) and various PTC clinicopathological features, as well as to evaluate the potential usefulness of these three potential oncogenic proteins in the prediction of aggressive PTC behavior.
Methods
ERα36, GRP78 and GRP94 protein expression in 218 primary PTC tissues and PTC-derived BCPAP cells was examined using immunohistochemistry, Western blotting and immunocytochemistry. The proliferative, invasive and migrative capacities of BCPAP cells in which the respective genes were either exogenously over-expressed or silenced were assessed using BrdU incorporation and Transwell assays, respectively.
Results
We found that ERα36, GRP78 and GRP94 protein expression was upregulated in the primary PTC tissues tested. We also found that ERα36, GRP78 and GRP94 expression modulation affected the proliferation, invasion and migration of PTC-derived BCPAP cells. A positive correlation and a positive feedback loop were noted between ERα36, GRP78 and GRP94 protein expression in the primary PTC tissues and in BCPAP cells, respectively. High ERα36 expression in combination with a high GRP78/ GRP94 expression was found to have a stronger correlation with extrathyroid extension (ETE), lymph node metastasis (LNM), distant metastasis (DM) and high TNM stage than high ERα36 expression in combination with either high GRP78 or high GRP94 expression (p = 0.028 for ETE, p = 0.002 for DM and p ≤ 0.001 for LNM and high TNM stage) or high ERα36 expression alone (p < 0.001 for ETE, LNM, DM and high TNM stage).
Conclusions
From our data we conclude that a concomitant high expression of ERα36, GRP78 and GRP94 is strongly associated with aggressive PTC behavior and may be used as a predictor for ETE, LNM, DM and high TNM stage.
Keywords: ERα36, GRP78, GRP94, Papillary thyroid cancer
Introduction
Clinical and epidemiological studies have shown that papillary thyroid cancer (PTC) accounts for 80% of all thyroid malignancies and that it is three times more common in women than in men, with the largest gender difference occurring during reproductive age and a decrease in incidence occurring after menopause [1, 2]. Since the elevated risk in women has also been reported to be associated with estrogen use [3, 4], it has been suggested this hormone may be involved in the development and progression of PTC, as has been shown for breast, endometrial and ovarian cancer [5, 6].
Estrogen receptor alpha36 (ERα36) is a 36 kDa variant of full-length ERα (66 kDa) that contains its DNA-binding, partial dimerization and ligand-binding domains, but lacks its transcription activation domains AF-1 and AF-2 [7, 8]. The C-terminal 27-amino acid domain of ERα36 is unique and replaces the last 138 amino acids encoded by exon 7 and 8 of the ERα gene. This unique sequence is thought to broaden the ligand-binding spectrum of ERα36 and, as such, to bind more ligands [8, 9]. Accordingly, it has been found that ERα36 can bind tamoxifen and fulvestrant, two ER inhibitors that are widely used in the clinic, that create an agonistic responses and that are involved in resistance to classical endocrine therapy in estrogen-related cancers [10–12]. ERα36 is mainly located in the cytoplasm, but may also be present at the cell surface where it mediates estrogen signaling through cross-talk with growth factor receptors and other signaling molecules (such as MAPK/ERK, PI3K/AKT and PKC) to promote cellular growth, invasion, migration and resistance to endocrine therapy [11, 13–16]. ERα36 over-expression has been observed in breast [16, 17], endometrial [18] and gastric tumors [19], and has been associated with malignancy, invasion, metastasis, treatment resistance and a poor prognosis. So far, a role of ERα36 expression in the development and progression of PTC has not been reported.
Glucose regulated proteins (GRPs) constitute a group of stress inducible endoplasmic reticulum-resident molecular chaperones participating in glycoprotein folding and facilitating the degradation of misfolded proteins [20, 21]. Interestingly, endoplasmic reticulum-resident GRPs have been found to translocate to the cell membrane in certain tumors and to be related to malignancy [21–23]. It has been found that the expression of GRP78 and GRP94, two prominent and well-studied GRPs, may be induced not only by tumor micro-environmental stress such as glucose and oxygen deprivation, but also by estrogen stimulation [24, 25]. GRP78 and GRP94 over-expression has also been associated with malignancy, invasion, metastasis, treatment resistance and a poor prognosis in several tumor types including breast, endometrial, ovarian, gastric and colorectal tumors [26–32]. Up to now, however, no study has dealt with the expression of GRP78 and GRP94 and its correlation with either ERα36 expression or aggressive PTC behavior.
Here, we simultaneously assessed ERα36, GRP78 and GRP94 expression in primary PTC tissues and PTC-derived cells, systematically assessed ERα36, GRP78 and GRP94 expression correlations with each other and with various clinicopathological features and evaluated the usefulness of these three putative oncogenic proteins for the prediction of aggressive PTC behavior.
Materials and methods
Case selection and tissue sample preparation
Tumor specimens were obtained from 218 PTC patients who underwent initial thyroidectomy in the Department of Surgery, the First Affiliated Hospital, Chongqing Medical University, between January 2010 and January 2015. At initial thyroidectomy, cervical lymph node dissection (CLND) was performed, tumor size was assessed, histologic subtype was established and extrathyroidal extension (ETE) and distant metastasis (DM) were evaluated. From the 218 cases, 135 were confirmed to be classic PTC, 36 confirmed to be follicular variants of PTC, 26 confirmed to be tall cell variants of PTC and 21 confirmed to be oncocytic variants of PTC. 48 patients were confirmed to have ETE, 105 patients confirmed to have lymph node metastasis (LNM), 61 patients confirmed to have DM, 85 patients with tumor sizes ≤ 2 cm, 81 patients with tumor sizes > 2 and ≤ 4 cm and 52 patients with tumor sizes > 4 cm. Of the patient cohort 54 were men and 164 were women; 60 patients were aged < 45 years and 158 were aged ≥ 45 years. According to TNM classification, there were 73 patients with stage I, 38 patients with stage II, 18 patients with stage III and 89 patients with stage IV. For statistical analysis, stage I and II were combined into low TNM stage (I-II) and stage III and IV were combined into high TNM stage (III-IV). Besides, benign thyroid tissue specimens were obtained from 156 patients with nodular hyperplasia. An additional 175 normal thyroid tissues were taken from the contralateral lobes of PTC patients to serve as controls. The study protocol was approved by the Ethics Committee of Chongqing Medical University and informed consent was obtained from all patients.
Tissue microarray and immunohistochemical staining
Tissue microarray construction and immunohistochemical staining were performed as reported before [33]. Rabbit polyclonal anti-ERα36 (1:100 dilution, BS1114; Cell Applications, USA), anti-GRP78 (1:50 dilution, ab188878; Abcam, USA) and anti-GRP94 (1:50 dilution, ab53075; Abcam, USA) antibodies were used as primary antibodies and biotinylated goat-anti-rabbit IgG (ZB-2010, Zhongshan Golden Bridge Biotechnology, China) as a secondary antibody (1:500 dilution).
IHC scoring
A semi-quantitative immunohistochemistry (IHC) scoring assessment was performed by two observers blinded to the diagnosis. IHC scores were based on staining intensity and percentage of positive cells. The intensities were assigned as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The percentages were assigned as 0 (< 5% positive cells), 1 (6–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells) and 4 (> 75% positive cells). Multiplication of the intensity and percentage scores resulted in the final IHC scores: 0 (negative), + (1–4), ++ (5–8) and +++ (9–12). For statistical analysis, a final IHC score of negative or + was defined as low expression group and a final IHC score of ++ or +++ as high expression group.
Cell culture and treatment
Human PTC-derived BCPAP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (GBICO Co. Ltd., Grand Island, NY, USA). The cells were incubated in phenol-red free and serum free medium for 48 h before stimulation with 17β-estradiol (E2) (Sigma, St Louis, MO, USA). Cell treatments were performed as described in the respective figure legends.
siRNA-mediated expression knockdown
For expression knockdown, specific siRNA vectors were constructed by cloning oligonucleotides targeting 3′UTR ERα36, GRP78 or GRP94 sequences into a pRNAT-U6.1/Neo expression vector (GenScript Corp. Piscataway, NJ, USA). These siRNA constructs were transfected into BCPAP cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions before treatment as indicated in the figure legends. A scrambled siRNA was transfected as control. The target sequences used were for ERα: 36 5′-GATGCCAATAGGTACTGAATTGATATCCGTTCAGTACCTATTGGCAT-3′, for GRP78: 5’-GCAAGAATTGAAATTGAGTTTCAAGAGAACTCAATTTCAATTCTTGC-3′, for GRP94: 5’-GAGGAAGAAGAAGAAGAAATTCAAGAGATTTCTTCTTCTTCTTCCTC-3′ and for scrambled siRNA: 5’-AACGTCACGTGTTAGATAATCGTGAAGTCAACTTAACAGTAATTCAC-3′.
Exogenous gene over-expression
Mammalian expression vectors for ERα36, GRP78 and GRP94 were constructed by cloning PCR-amplified fragments corresponding to the respective open-reading frame sequences into a pcDNA3.1 expression vector (Invitrogen). These expression constructs were transfected into BCPAP cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions before treatment as indicated in the figure legends.
Protein extraction and Western blotting
Cells were scraped from the culture plates, washed with ice-cold PBS and gently lysed for 30 min in 5 volumes of ice-cold RIPA protein extraction buffer supplemented with a Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) at a ratio of 1000:1. The resulting cell lysates were centrifuged at 10000×g for 10 min at 4 °C to obtain supernatants (whole cell lysates). The protein content of the cell lysates was quantified using a Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, Hercules, CA, USA), after which 30 μg total protein was loaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gels, separated and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes were incubated with primary antibodies diluted in TBST (containing 0.1% Tween 20 and 2% BSA) overnight at 4 °C. Subsequently, the membranes were washed and incubated with appropriate secondary antibodies, after which the protein bands were detected using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). Finally, densitometric analysis was performed using the TINA version 2.09 program package. β-actin was used as a control for equal loading.
IHC of cultured cells
Cells were seeded on coverslips and treated as indicated in the figure legends. Next, the cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 20 min. After blocking with 1% Bovine Serum Albumin (BSA) for 1 h, the cells were incubated with mouse monoclonal antibodies directed against GRP78 (1:50, ab181499; Abcam, USA) and GRP94 (1:50, ab210960, Abcam, USA) and a rabbit polyclonal antibody directed against ERα36 (1:100 dilution, CY1109; Cell Applications, USA) overnight at 4 °C. The next day, the cells were incubated with Alexa Fluor 594-Conjugated AffiniPure Goat Anti-Mouse IgG (ZF-0513, Zhongshan Golden Bridge Biotechnology, China) and Alexa Fluor 488-Conjugated AffiniPure Goat Anti-Rabbit IgG (ZF-0511, Zhongshan Golden Bridge Biotechnology, China) for 1 h, respectively. Images were acquired by confocal microscopy (LSM Meta 700, Carl Zeiss, Oberkochen, Germany) and analyzed using LSM Image Browser software.
Cell proliferation assay
Cell proliferation was assayed using a BrdU incorporation Colorimetric ELISA kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
In vitro migration and invasion assays
In vitro migration and invasion capacities were assessed using Transwell assays (24-well insert, pore size 8 μm; Corning Inc., Corning, NY, USA). Briefly, 3 × 104 quiescent cells were resuspended in phenol-red free and serum-free medium and placed in the top chambers. The lower chambers were filled with phenol-red free and serum-free medium supplemented with E2 for 48 h. For the invasion assay, the inserts were pre-coated with extracellular matrix gel (BD Biosciences, Bedford, MA, USA). At the end of the experiments, MTT was added for another 4 h. After this, the cells from the top of the Transwell chambers were removed using cotton swabs (residual cells). The cotton swabs containing residual cells and the Transwell chamber migrated or invaded cells were placed in 24-well plates containing 400 μl DMSO. After 1 h of gentle shaking, 100 μl samples were taken and the absorbance was measured at 570 nm using an ELISA plate reader. The percentage of migrated or invaded cells was calculated as: percentage of migrated or invaded cells = A/(A + B) × 100, where A represents the absorbance of the migrated or invaded cells and B the absorbance of the residual cells.
Statistical analysis
Statistical analyses were performed using SPSS 18.0 statistical software. Data are presented as percentages and mean plus standard deviation, according to distribution. Significance was assessed using χ2, Spearman rank and Student’s t-tests, as appropriate, to compare groups. A p value < 0.05 was considered to be statistically significant.
Results
ERα36, GRP78 and GRP94 expression levels are upregulated in PTC tissues
ERα36, GRP78 and GRP94 protein expression levels in PTC, nodular hyperplasia and normal thyroid tissues were examined by IHC staining. For all three proteins immunoreactivities were observed in both the cytoplasm and membranes of the cells. In normal thyroid and nodular hyperplasia tissues almost no follicular cells were found to stain positive for either ERα36 (Fig. 1a and d), GRP78 (Fig. 1b and e) or GRP94 (Fig. 1c and f). In the PTC tissues, however, we found that several cases contained tumor cells with moderate ERα36 (Fig. 1g), GRP78 (Fig. 1h) and GRP94 (Fig. 1i) staining, indicating low expression of these three proteins, whereas other cases contained tumor cells with strong ERα36 (Fig. 1j), GRP78 (Fig. 1k) and GRP94 (Fig. 1l) staining, indicating high expression of these three proteins. Similar to normal thyroid tissues, we found that the majority of nodular hyperplasia tissues were negative or had an IHC score 1 for ERα36, GRP78 and GRP94, whereas none of the cases exhibited a high expression (IHC score ≥ 5) of either of the three proteins (Table 1). The majority of the PTC tissues exhibited IHC scores ≥ 2 for all three proteins, whereas high IHC scores (≥ 5) were noted in 112 (51.4%), 129 (59.2%) and 120 (55.0%) of the cases for ERα36, GRP78 and GRP94, respectively. The differences observed in ERα36, GRP78 and GRP94 protein expression between the PTC tissues at one hand and the normal thyroid and nodular hyperplasia tissues at the other hand were found to be statistically significant (p < 0.001) (Table 2).
Fig. 1.
ERα36, GRP78 and GRP94 IHC staining. The first and second rows (a-f) show examples of almost no follicular cells with positive ERα36, GRP78 and GRP94 staining in normal thyroid tissues (a-c) and nodular hyperplasia tissues (d-f). The third and fourth rows (g-l) show examples of low (g-i) and high (j-l) ERα36, GRP78 and GRP94 expression in PTC tissues. All pictures represent high-power fields (400×)
Table 1.
IHC expression scores of ERα36, GRP78 and GRP94 in 218 PTC, 156 nodular hyperplasia and 175 normal thyroid tissues
| Score | ERα36 | GRP78 | GRP94 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Normal thyroid tissues n |
Nodular hyperplasia tissues n |
PTC tissues n |
Normal thyroid tissues n |
Nodular hyperplasia tissues n |
PTC tissues n |
Normal thyroid tissues n |
Nodular hyperplasia tissues n |
PTC tissues n |
|
| 0 | |||||||||
| Negative | 143 | 103 | 9 | 136 | 98 | 6 | 148 | 100 | 8 |
| + | |||||||||
| 1 | 28 | 51 | 14 | 34 | 49 | 12 | 24 | 52 | 15 |
| 2 | 4 | 2 | 20 | 5 | 9 | 17 | 3 | 4 | 18 |
| 3 | 0 | 0 | 31 | 0 | 0 | 26 | 0 | 0 | 28 |
| 4 | 0 | 0 | 32 | 0 | 0 | 28 | 0 | 0 | 29 |
| ++ | |||||||||
| 6 | 0 | 0 | 32 | 0 | 0 | 35 | 0 | 0 | 35 |
| 8 | 0 | 0 | 34 | 0 | 0 | 38 | 0 | 0 | 34 |
| +++ | |||||||||
| 9 | 0 | 0 | 30 | 0 | 0 | 36 | 0 | 0 | 32 |
| 12 | 0 | 0 | 16 | 0 | 0 | 20 | 0 | 0 | 19 |
The IHC expression scores were determined by multiplication of percentage and intensity scores
Table 2.
Correlation of ERα36, GRP78 and GRP94 protein expression with clinicopathological characteristics in 218 PTC cases
| Characteristics | Case (n) | ERα36 | GRP78 | GRP94 | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Low | High | p-value | Low | High | p-value | Low | High | p-value | ||
| Tissue type | ||||||||||
| Normal thyroid tissue | 175 | 175 | 0 | 175 | 0 | 175 | 0 | |||
| Nodular hyperplasia | 156 | 156 | 0 | – | 156 | 0 | – | 156 | 0 | – |
| PTC | 218 | 106 | 112 | < 0.001a | 89 | 129 | < 0.001a | 98 | 120 | < 0.001a |
| < 0.001b | < 0.001b | < 0.001b | ||||||||
| Classic PTC | 135 | 73 | 62 | 0.230 | 63 | 72 | 0.168 | 68 | 67 | 0.193 |
| Follicular Variant of PTC | 36 | 15 | 21 | 11 | 25 | 12 | 24 | |||
| Tall Cell Variant of PTC | 26 | 10 | 16 | 8 | 18 | 9 | 17 | |||
| Oncocytic Variant of PTC | 21 | 8 | 13 | 7 | 14 | 9 | 12 | |||
| Age (years) | ||||||||||
| < 45 | 60 | 34 | 26 | 0.143 | 28 | 32 | 0.280 | 31 | 29 | 0.220 |
| ≥ 45 | 158 | 72 | 86 | 61 | 97 | 67 | 91 | |||
| Gender | ||||||||||
| Male | 54 | 30 | 24 | 0.240 | 18 | 36 | 0.197 | 20 | 34 | 0.178 |
| Female | 164 | 76 | 88 | 71 | 93 | 78 | 86 | |||
| Tumor size (cm) | ||||||||||
| T1 ≤ 2 | 85 | 48 | 37 | 0.150 | 39 | 46 | 0.124 | 44 | 41 | 0.146 |
| 2 < T2 ≤ 4 | 81 | 37 | 44 | 35 | 46 | 36 | 45 | |||
| T3 > 4 | 52 | 21 | 31 | 15 | 37 | 18 | 34 | |||
| Extrothyroid extension (ETE) | ||||||||||
| Absent | 170 | 106 | 64 | < 0.001 | 89 | 81 | < 0.001 | 98 | 72 | < 0.001 |
| Present | 48 | 0 | 48 | 0 | 48 | 0 | 48 | |||
| Lymph node metastasis (LNM) | ||||||||||
| Absent | 113 | 86 | 27 | < 0.001 | 72 | 41 | < 0.001 | 80 | 33 | < 0.001 |
| Present | 105 | 20 | 85 | 17 | 88 | 18 | 87 | |||
| Distant metastasis (DM) | ||||||||||
| Absent | 157 | 105 | 52 | < 0.001 | 89 | 68 | < 0.001 | 98 | 59 | < 0.001 |
| Present | 61 | 1 | 60 | 0 | 61 | 0 | 61 | |||
| TNM stage | ||||||||||
| I-II | 111 | 84 | 27 | < 0.001 | 72 | 39 | < 0.001 | 79 | 32 | < 0.001 |
| III-IV | 107 | 22 | 85 | 17 | 90 | 19 | 88 | |||
P-values were derived using a χ2 test to compare ERα36, GRP78 and GRP94 expression between subgroups defined by clinicopathological characteristics; a significant difference between PTC and normal thyroid tissues; b significant difference between PTC and nodular hyperplasia tissues. p < 0.05 was considered to be statistically significant
High ERα36, GRP78 and GRP94 expression correlates with aggressive PTC behavior
To examine whether high ERα36, GRP78 and GRP94 expression correlates with aggressive PTC behavior, we systematically assessed correlations between expression scores and various clinicopathological characteristics of the PTC patients. We found that ERα36 protein expression significantly correlated with ETE (p < 0.001), LNM (p < 0.001), DM (p < 0.001) and TNM stage (p < 0.001), i.e., PTC patients with ETE, LNM, DM and a high TNM stage (III-IV) exhibited high ERα36 protein expression levels (Table 2). In contrast, no statistically significant differences were found in ERα36 protein expression between PTC patients with different histologic subtypes (p = 0.230), between older (≥ 45) and younger (< 45) patients (p = 0.143), between male and female patients (p = 0.240) and between patients with large or small tumor sizes (p = 0.150). In the case of GRP78 and GRP94, no correlation was found between their expression and histologic subtype, age, gender and tumor size (p = 0.168, 0.280, 0.197, 0.124 for GRP78 and p = 0.193, 0.220, 0.178, 0.146 for GRP94, respectively). In contrast, we found that GRP78 and GRP94 protein expression significantly correlated with ETE (p < 0.001), LNM (p < 0.001), DM (p < 0.001) and TNM stage (p < 0.001). PTC patients with ETE, LNM, DM and a high TNM stage (III-IV) exhibited higher expression levels of the two proteins than those with a low TNM stage (I-II) and those without ETE, LNM and DM. From these results we conclude that high ERα36, GRP78 and GRP94 expression correlates with aggressive PTC behavior, including ETE, LNM, DM and high TNM stage.
ERα36, GRP78 and GRP94 protein expression levels are correlated in PTC tissues
To examine whether the ERα36, GRP78 and GRP94 protein expression levels are correlated with each other in PTC tissues, we applied a Spearman rank test. By doing so, we found that 79/218 of the cases showed a high ERα36 and GRP78 expression and that 56/218 of the cases showed a low expression of these two proteins (Table 3). This correlation was found to be statistically significant (rs = 0.238, p < 0.001). Similarly, a statistically significant correlation was found between ERα36 and GRP94 expression (rs = 0.320, p < 0.001), i.e., 79/218 PTC tissues showed a high ERα36 and GRP94 expression. In addition, we found a high GRP78 and GRP94 expression in 80/218 PTC tissues. Also this positive correlation was found to be statistically significant (rs = 0.169, p = 0.013). From these results we conclude that the ERα36, GRP78 and GRP94 protein expression levels are correlated with each other in PTC tissues.
Table 3.
Correlations of ERα36, GRP78 and GRP94 protein expression levels between each other in 218 PTC tissues
| Proteins | ERα36 | GRP94 | ||||||
|---|---|---|---|---|---|---|---|---|
| Low | High | r s | p-value | Low | High | r s | p-value | |
| GRP78 | ||||||||
| Low | 56 | 33 | 0.238 | < 0.001 | 49 | 40 | 0.169 | 0.013 |
| High | 50 | 79 | 49 | 80 | ||||
| GRP94 | ||||||||
| Low | 65 | 33 | 0.320 | < 0.001 | ||||
| High | 41 | 79 | ||||||
P-values from Spearman rank test; ERα36, GRP78 and GRP94 were tested pair-wise; p < 0.05 was considered to be statistically significant
E2 upregulates ERα36, GRP78 and GRP94 expression and a positive feedback loop exists between ERα36 and GRP78/GRP94 expression
Since we found that the ERα36, GRP78 and GRP94 protein expression levels are correlated with each other in PTC tissues, and since 17β-estradiol (E2) is known to be involved in the development and progression of PTC [1–4], we set out to perform in vitro assays to determine whether the ERα36, GRP78 and GRP9 protein expression levels are upregulated by E2 and whether a positive feedback loop exists between ERα36, GRP78 and GRP94 expression in human BCPAP cells. Using Western blotting, we found that the ERα36, GRP78 and GRP94 protein expression levels are upregulated by E2 (Fig. 2). In addition, we found that the E2 upregulated GRP78 and GRP94 protein expression levels were attenuated by siRNA-mediated ERα36 silencing and augmented by exogenous ERα36 over-expression. Reciprocally, we found that the E2 upregulated ERα36 protein expression level was attenuated by siRNA-mediated GRP78 and GRP94 silencing and augmented by exogenous GRP78 and GRP94 over-expression. These results indicate that positive feedback loops exist between ERα36 and GRP78/GRP94 protein expression. Through double immunofluorescent staining, we observed co-localizations of ERα36 and GRP78, and of ERα36 and GRP94 in the both the cytoplasm and membranes of BCPAP cells (Fig. 3).
Fig. 2.
Positive feedback loop between ERα36 and GRP78/GRP94 expression in BCPAP cells. Quiescent BCPAP cells were transfected with ERα36, GRP78 and GRP94 siRNAs or expression vectors and stimulated with E2 for 24 h, after which ERα36, GRP78 and GRP94 protein expression levels were assessed by Western blotting. β-actin was used as loading control (a, c, e, g). Bar diagrams indicate relative ERα36, GRP78 and GRP94 protein levels (b, d, f, h). The data represent the means of three independent experiments. * p < 0.05, compared to vehicle (Veh) control. # p < 0.05, compared to E2 treatment alone. P-values were derived using Student’s t-test
Fig. 3.
Co-localization of ERα36 with GRP78 and GRP94 in BCPAP cells. Quiescent BCPAP cells were exposed to E2 for 24 h and then assayed by double immunofluorescence staining. (a, d) ERα36 staining with green fluorescence and (b, e) GRP78 and GRP94 staining with red fluorescence. (c, f) Merged images of ERα36 and GRP78 staining and ERα36 and GRP94 staining. Yellow fluorescence indicates overlap. Figures are representatives of three independent experiments
E2 induced upregulation of ERα36, GRP78 and GRP94 enhances the proliferation, invasion and migration of PTC cells
Since we found that high ERα36, GRP78 and GRP94 expression levels were correlated with aggressive PTC behavior, we set out to perform in vitro assays to assess whether E2 induced upregulation of ERα36, GRP78 and GRP9 enhances the proliferation, invasion and migration of BCPAP cells. To this end, ERα36, GRP78 and GRP94 siRNA and over-expression vectors were used. We found that E2 enhanced the proliferation, invasion and migration of BCPAP cells. These effects were attenuated by siRNA-mediated silencing of ERα36, GRP78 and GRP94 and augmented by exogenous over-expression of ERα36, GRP78 and GRP94. Control scrambled siRNAs and empty expression vectors had no effects on the E2 induced proliferation, invasion and migration of BCPAP cells (Fig. 4). These results indicate that E2 induced upregulation of ERα36, GRP78 and GRP94 is involved in enhancing the proliferation, invasion and migration of PTC cells.
Fig. 4.
E2 induced ERα36, GRP78 and GRP94 upregulation enhances the proliferation, invasion and migration of BCPAP cells. Quiescent BCPAP cells were stimulated with E2 for 48 h, after which proliferation was assessed by BrdU incorporation, and invasion and migration by Transwell assays. Quiescent BCPAP cells were also transfected with siRNAs, over-expression vectors, scrambled siRNAs and empty vectors prior to stimulation with E2. (a) Effects of ERα36, GRP78 and GRP94 expression on the proliferation of BAPAP cells. (b) Effects of ERα36, GRP78 and GRP94 expression on the migration of BAPAP cells. (c) Effects of ERα36, GRP78 and GRP94 expression on the invasion of BAPAP cells. The data represent the means of three independent experiments in triplicate. * p < 0.05, compared to vehicle (Veh) control. # p < 0.05, compared to E2 treatment alone. P-values were derived using Student’s t-test
Concomitant high ERα36, GRP78 and GRP94 expression is associated with ETE, LNM, DM and high TNM stage
Based on the above observations (i) that the ERα36, GRP78 and GRP94 expression levels are correlated, (ii) that positive feedback loops exist between ERα36 and GRP78 and between ERα36 and GRP94 expression, (iii) that upregulation of these three proteins enhances the proliferation, invasion and migration of PTC cells and (iv) that PTC patients with ETE, LNM, DM and a high TNM stage exhibit higher expression levels of these proteins than those with a low TNM stage and without ETE, LNM and DM, we next set out to assess associations between ETE, LNM, DM and high TNM stage and concomitant high ERα36, GRP78 and GRP94 expression levels in our patient cohort. We found that 76 PTC patients showed concomitant high expression levels of the three proteins, including 76 patients with a high TNM stage, 60 patients with DM and 16 patients without DM, 75 patients with LNM and one without LNM and 48 patients with ETE and 28 without ETE, respectively (Table 4). We found that the incidence of high TNM stage, DM, LNM and ETE was significantly higher in patients with a high ERα36 expression combined with both a high GRP78 and a high GRP94 expression (100% for high TNM stage, 78.9% for DM, 98.7% for LNM and 63.2% for ETE, respectively) than in patients with a high ERα36 expression combined with either a high GRP78 or a high GRP94 expression (66.7% and 33.3% for high TNM stage and LNM and 0% for DM and ETE, respectively), in patients with a high ERα36 expression only (20.0% for high TNM stage, 23.3% for LNM and 0% for DM and ETE, respectively) and in patients without a high expression of any of the three proteins (5.3% for high TNM stage, 0% for DM, LNM and ETE, respectively). The concomitant high expression of all the three proteins was significantly associated with a high TNM stage, DM, LNM and ETE compared to cases without such a concomitant expression (p < 0.001). In Fig. 5 a-c results of a representative PTC patient with TNM stage I and without ETE, LNM and DM exhibiting a low expression of ERα36, GRP78 and GRP94 are shown, whereas in Fig. 5d-f results of a representative PTC patient with TNM stage IV, ETE, LNM and DM exhibiting a high expression of all the three proteins are shown. When concomitant high expression of all the three proteins was used as a predictive indicator for high TNM stage, DM, LNM and ETE, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy were found to be 71.0%, 100%, 100%, 78.2%, 85.8% for high TNM stage, 98.4, 89.8, 78.9, 99.3, 92.2 for DM, 71.4%, 99.1%, 98.7%, 78.9%, 85.8% for LNM and 100.0%, 83.5%, 63.2%, 100.0%, 87.2% for ETE, respectively.
Table 4.
Correlation of concomitant ERα36, GRP78 and GRP94 expression with ETE, LNM, DM and TNM stage in PTC patients
| ETE | LNM | DM | TNM stage | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Absent n (%) | Present n (%) | p- value | Absent n (%) | Present n (%) | p- value | Absent n (%) | Present n (%) | p- value | I-II n (%) | III-IV n (%) | p- value | |
| Expression information | ||||||||||||
| (1) All of ERα36/GRP78/GRP94 low expression | 19 (100.0) | 0 (0) | < 0.001a | 19 (100.0) | 0 (0) | < 0.001a | 19 (100.0) | 0 (0) | < 0.001a | 18 (94.7) | 1 (5.3) | < 0.001a |
| (2) ERα36 high expression; GRP78/GRP94 low expression | 30 (100.0) | 0 (0) | < 0.001b | 23 (76.7) | 7 (23.3) | < 0.001b | 30 (100.0) | 0 (0) | < 0.001b | 24 (80.0) | 6 (20.0) | < 0.001b |
| (3) GRP78 high expression; ERα36/ GRP94 low expression | 46 (100.0) | 0 (0) | < 0.001c | 37 (80.4) | 9 (19.6) | < 0.001c | 46 (100.0) | 0 (0) | < 0.001c | 36 (78.2) | 10 (21.7) | < 0.001c |
| (4) GRP94 high expression; ERα36/GRP78 low expression | 37 (100.0) | 0 (0) | < 0.001d | 28 (75.7) | 9 (24.3) | < 0.001d | 37 (100.0) | 0 (0) | < 0.001d | 28 (75.7) | 9 (24.3) | < 0.001d |
| (5) ERα36/GRP78 high expression; GRP94 low expression | 3 (100.0) | 0 (0) | 0.028 e | 1 (33.3) | 2 (66.7) | 0.001e | 3 (100.0) | 0 (0) | 0.002 e | 1 (33.3) | 2 (66.7) | < 0.001e |
| (6) ERα36/GRP94 high expression; GRP78 low expression | 3 (100.0) | 0 (0) | 0.028 f | 2 (66.7) | 1 (33.3) | < 0.001f | 3 (100.0) | 0 (0) | 0.002 f | 2 (66.7) | 1 (33.3) | < 0.001f |
| (7) GRP78/GRP94 high expression; ERα36 low expression | 4 (100.0) | 0 (0) | 0.012 g | 2 (50.0) | 2 (50.0) | < 0.001g | 3 (100.0) | 1 (0) | 0.013 g | 2 (50.0) | 2 (50.0) | < 0.001g |
| (8) All of ERα36/GRP78/GRP94 high expression | 28 (36.8) | 48 (63.2) | < 0.001h | 1 (1.3) | 75 (98.7) | < 0.001h | 16 (21.1) | 60 (78.9) | < 0.001h | 0 (0) | 76 (100) | < 0.001h |
| Evaluation index (Concomitant high expression of ERα36/GRP78/GRP94) | Rate (%) | Rate (%) | Rate (%) | Rate (%) | ||||||||
| Sensitivity | 100.0 | 71.4 | 98.4 | 71.0 | ||||||||
| Specificity | 83.5 | 99.1 | 89.8 | 100 | ||||||||
| Positive predictive value(PPV) | 63.2 | 98.7 | 78.9 | 100 | ||||||||
| Negative predictive value(NPV) | 100.0 | 78.9 | 99.3 | 78.2 | ||||||||
| Diagnostic accuracy | 87.2 | 85.8 | 92.2 | 85.8 | ||||||||
Correlation of concomitant ERα36, GRP78 and GRP94 expression with ETE, LNM, DM and TNM stage was measured by χ2 test; a significant difference between group (1) and group (8); b significant difference between group (2) and group (8); c significant difference between group (3) and group (8); d significant difference between group (4) and group (8); e significant difference between group (5) and group (8); f significant difference between group (6) and group (8); g significant difference between group (7) and group (8); h significant difference between groups with and without concomitant high expression of all the three proteins. p < 0.05 was considered to be statistically significant
Fig. 5.
Association of concomitant high ERα36, GRP78 and GRP94 expression with ETE, LNM, DM and high TNM stage. In the first row immunostaining of a representative PTC patient is depicted with TNM stage I and without ETE, LNM and DM showing low ERα36 (a), GRP78 (b) and GRP94 (c) expression. In the second row immunostaining of a representative PTC patient is depicted with TNM stage IV, ETE, LNM and DM showing high ERα36 (d), GRP78 (e) and GRP94 (f) expression. All the pictures represent high-power fields (400×)
Discussion
Clinical and epidemiological studies have suggested that estrogen may be involved in the development and progression of PTC [1–4], as has amply been demonstrated in breast, endometrial and ovarian carcinomas [5]. It is widely accepted that estrogen acts through interaction with two estrogen receptors, ERα and ERβ, which belong to the nuclear steroid hormone receptor family and function indisputably as transcription factors that induce estrogen-dependent gene transactivation [34]. In the past, ERα36 has been identified as a new member of the ER family and found to be located in the cytoplasm as well as on the cell surface where it mediates non-genomic estrogen signaling through cross-talk with growth factor receptors and other signaling molecules (such as MAPK/ERK, PI3K/AKT and PKC) to promote cellular growth, invasion and migration, and resistance to endocrine therapy [11, 13–16]. GRP78 and GRP94 are two well-studied stress-inducible endoplasmatic reticulum-resident molecular chaperones that can be induced not only by tumor microenvironmental stress such as glucose and oxygen deprivation, but also by estrogen stimulation [24, 25]. Interestingly, it has been found that the endoplasmic reticulum-resident proteins GRP78 and GRP94 translocate to the cell membrane in certain tumor cells and that this translocation is related to malignancy [21–23]. More recently, it has been found that ERα36, GRP78 and GRP94 are over-expressed in breast, endometrial, ovarian, gastric and colorectal tumors, and that this over-expression is associated with malignancy, invasion, metastasis, drug resistance and poor prognosis [16–19, 26–32]. As of yet, however, no study has been dealt with the expression of ERα36, GRP78 and GRP94 and its correlation with clinicopathological features in PTC patients. We assessed ERα36, GRP78 and GRP94 protein expression in primary PTC tissues as well as in nodular hyperplasia and normal thyroid tissues using immunohistochemistry. We found that none of the normal thyroid and nodular hyperplasia tissues showed high ERα36, GRP78 or GRP94 protein expression levels. Conversely, we found high ERα36, GRP78 and GRP94 protein expression levels in 51.4%, 59.2% and 55.0% of the PTC tissues tested, respectively. The differences in ERα36, GRP78 and GRP94 expression between PTC and normal thyroid tissues as well nodular hyperplasia tissues were statistically significant (p < 0.001). We also found that the ERα36, GRP78 and GRP94 protein expression levels were significantly correlated with PTC aggressiveness, including ETE, LNM, DM and a high TNM stage, whereas no correlation was found between the expression of these three proteins and histologic subtype, age, gender and tumor size. PTC patients with ETE, LNM, DM and a high TNM stage (III-IV) exhibited higher ERα36, GRP78 and GRP94 protein expression levels than those with a low TNM stage (I-II) and without ETE, LNM, DM. Subsequent in vitro assays showed that 17β-estradiol (E2)-induced ERα36, GRP78 and GRP94 upregulation resulted in an enhanced proliferation, invasion and migration of PTC-derived cells. These results are concordant with previous results in other tumor types such as breast, endometrial, ovarian, gastric and colorectal tumors [16–19, 26–32], indicating that E2-induced ERα36, GRP78 and GRP94 upregulation may play a role in the progression and metastasis of PTC.
Recent studies have shown that, as molecular chaperones, GRP78 and GRP94 may interact with ERα and ERα36 and, by doing so, upregulate ERα and ERα36 expression levels by preventing their ubiquitination and proteasomal degradation [35, 36]. Accordingly, we noted a significant positive correlation between ERα36, GRP78 and GRP94 expression in PTC tissues. A significant positive correlation was also noted between GRP78 and GRP94 expression. Additional in vitro assays showed that E2 could upregulate ERα36, GRP78 and GRP94 expression in PTC-derived cells. A positive feedback loop was uncovered between ERα36 and GRP78 expression and between ERα36 and GRP94 expression. Additionally, we observed co-localization of ERα36 and GRP78 and ERα36 and GRP94 in the cytoplasm and cell membranes of PTC-derived cells. The existence of these positive correlations and feedback loops is supported by the finding that estrogen stimulation may lead to upregulation and activation of ERα36 which, in turn, may boost the MAPK/ERK and PI3K/AKT signal pathways and promote tumor progression and metastasis through GRP78 and GRP94 upregulation [37–39]. Conversely, as molecular chaperones, GRP78 and GRP94 may interact with ERα36 and upregulate its expression level by preventing its ubiquitination and proteasomal degradation. We also found that a high ERα36 expression combined with both a high GRP78 and a high GRP94 expression strongly correlated with ETE, LNM, DM and a high TNM stage. As such, this combined high expression may serve as a predictor of aggressive PTC behavior.
In summary, we found a positive correlation and a positive feedback loop between ERα36, GRP78 and GRP94 expression in primary PTC tissues and PTC-derived cells. A high ERα36, GRP78 and GRP94 expression may serve as an ETE, LNM, DM and high TNM stage predictor in PTC patients.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 81272937).
Compliance with ethical standards
Conflict of interest
There was no conflict of interest to declare.
Footnotes
Yu-Jie Dai and Yi-Bo Qiu contributed equally to this work.
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